Despite being the leading cause of cancer-related childhood mortality, pediatric gliomas have been relatively understudied, and the repurposing of immunotherapies has not been successful. Whole-transcriptome sequencing, single-cell sequencing, and sequential multiplex immunofluorescence were used to identify an immunotherapeutic strategy that could be applied to multiple preclinical glioma models. MAPK-driven pediatric gliomas have a higher IFN signature relative to other molecular subgroups. Single-cell sequencing identified an activated and cytotoxic microglia (MG) population designated MG-Act in BRAF-fused, MAPK-activated pilocytic astrocytoma (PA), but not in high-grade gliomas or normal brain. T cell immunoglobulin and mucin domain 3 (TIM3) was expressed on MG-Act and on the myeloid cells lining the tumor vasculature but not normal brain vasculature. TIM3 expression became upregulated on immune cells in the PA microenvironment, and anti-TIM3 reprogrammed ex vivo immune cells from human PAs to a proinflammatory cytotoxic phenotype. In a genetically engineered murine model of MAPK-driven, low-grade gliomas, anti-TIM3 treatment increased median survival over IgG- and anti–PD-1–treated mice. Single-cell RNA-Seq data during the therapeutic window of anti-TIM3 revealed enrichment of the MG-Act population. The therapeutic activity of anti-TIM3 was abrogated in mice on the CX3CR1 MG–KO background. These data support the use of anti-TIM3 in clinical trials of pediatric low-grade, MAPK-driven gliomas.
Shashwat Tripathi, Hinda Najem, Corey Dussold, Sebastian Pacheco, Ruochen Du, Moloud Sooreshjani, Lisa Hurley, James P. Chandler, Roger Stupp, Adam M. Sonabend, Craig M. Horbinski, Rimas V. Lukas, Joanne Xiu, Giselle Lopez, Theodore P. Nicolaides, Valerie Brown, Nitin R. Wadhwani, Sandi K. Lam, Charles David James, Ganesh Rao, Maria G. Castro, Amy B. Heimberger, Michael DeCuypere
The burden of senescent hepatocytes correlates with the severity of metabolic dysfunction–associated steatotic liver disease (MASLD), but the mechanisms driving senescence and how it exacerbates MASLD are poorly understood. Hepatocytes experience lipotoxicity and become senescent when Smoothened (Smo) is deleted to disrupt Hedgehog signaling. We aimed to determine whether the secretomes of Smo-deficient hepatocytes perpetuate senescence to drive MASLD progression. RNA-Seq analysis of liver samples from human and murine cohorts with MASLD confirmed that hepatocyte populations in MASLD livers were depleted of Smo+ cells and enriched with senescent cells. When fed a choline-deficient, amino acid–restricted high-fat diet (CDA-HFD) to induce MASLD, Smo– mice had lower antioxidant markers and developed worse DNA damage, senescence, steatohepatitis, and fibrosis than did Smo+ mice. Sera and hepatocyte-conditioned medium from Smo– mice were depleted of thymidine phosphorylase (TP), a protein that maintains mitochondrial fitness. Treating Smo– hepatocytes with TP reduced senescence and lipotoxicity, whereas inhibiting TP in Smo+ hepatocytes had the opposite effect and exacerbated hepatocyte senescence, steatohepatitis, and fibrosis in CDA-HFD–fed mice. We conclude that inhibition of Hedgehog signaling in hepatocytes promoted MASLD by suppressing hepatocyte production of proteins that prevent lipotoxicity and senescence.
Ji Hye Jun, Kuo Du, Rajesh Kumar Dutta, Raquel Maeso-Diaz, Seh Hoon Oh, Liuyang Wang, Guannan Gao, Ana Ferreira, Jon Hill, Steven S. Pullen, Anna Mae Diehl
Patients with autism spectrum disorder (ASD) frequently experience sleep disturbance. Genetic mutations in the neuroligin 3 (NLG3) gene are highly correlative with ASD and sleep disturbance. However, the cellular and neural circuit bases of this correlation remain elusive. Here, we found that the conditional knockout of Nlg3 (Nlg3-CKO) in the medial septum (MS) impairs social memory and reduces sleep. Nlg3 CKO in the MS caused hyperactivity of MSGABA neurons during social avoidance and wakefulness. Activation of MSGABA neurons induced social memory deficits and sleep loss in C57BL/6J mice. In contrast, inactivation of these neurons ameliorated social memory deficits and sleep loss in Nlg3-CKO mice. Sleep deprivation led to social memory deficits, while social isolation caused sleep loss, both resulting in a reduction in NLG3 expression and an increase in activity of GABAergic neurons in the MS from C57BL/6J mice. Furthermore, MSGABA-innervated CA2 neurons specifically regulated social memory without impacting sleep, whereas MSGABA-innervating neurons in the preoptic area selectively controlled sleep without affecting social behavior. Together, these findings demonstrate that the hyperactive MSGABA neurons impair social memory and disrupt sleep resulting from Nlg3 CKO in the MS, and achieve the modality specificity through their divergent downstream targets.
Haiyan Sun, Yu Shen, Pengtao Ni, Xin Liu, Yan Li, Zhentong Qiu, Jiawen Su, Yihan Wang, Miao Wu, Xiangxi Kong, Jun-Li Cao, Wei Xie, Shuming An
Cardiac mononuclear phagocytic cells (Cardiac MPCs) participate in maintaining homeostasis and orchestrating cardiac responses upon injury. However, the function of specific MPC subtypes and the related cell fate commitment mechanisms remain elusive in regenerative and nonregenerative hearts due to their cellular heterogeneities. Using spatiotemporal single-cell epigenomic analysis of cardiac MPCs in regenerative (P1) and nonregenerative (P10) mouse hearts after injury, we found that P1 hearts accumulate reparative Arg1+ macrophages, while proinflammatory S100a9+Ly6c+ monocytes are uniquely abundant during nonregenerative remodeling. Moreover, blocking chemokine CXCR2 to inhibit the specification of the S100a9+Ly6c+-biased inflammatory fate in P10 hearts resulted in elevated wound repair responses and marked improvements in cardiac function after injury. Single-cell RNA-Seq further confirmed an increased Arg1+ macrophage subpopulation after CXCR2 blockade, which was accomplished by increased expression of wound repair–related genes and reduced expression of proinflammatory genes. Collectively, our findings provide instructive insights into the molecular mechanisms underlying the function and fate specification of heterogeneous MPCs during cardiac repair and identify potential therapeutic targets for myocardial infarction.
Mingzhu Fu, Shengtao Jia, Longhui Xu, Xin Li, Yufang Lv, Yulong Zhong, Shanshan Ai
Variants of the G protein–coupled receptor 75 (GPR75) are associated with a lower BMI in large-scale human exome-sequencing studies. However, how GPR75 regulates body weight remains poorly understood. Using random germline mutagenesis in mice, we identified a missense allele (Thinner) of Gpr75 that resulted in a lean phenotype and verified the decreased body weight and fat weight in Gpr75-knockout (Gpr75–/–) mice. Gpr75–/– mice displayed reduced food intake under high-fat diet (HFD) feeding, and pair-feeding normalized their body weight. The endogenous GPR75 protein was exclusively expressed in the brains of 3xFlag-tagged Gpr75-knockin (3xFlag-Gpr75) mice, with consistent expression across different brain regions. GPR75 interacted with Gαq to activate various signaling pathways after HFD feeding. Additionally, GPR75 was localized in the primary cilia of hypothalamic cells, whereas the Thinner mutation (L144P) and human GPR75 variants in individuals with a lower BMI failed to localize in the cilia. Loss of GPR75 selectively inhibited weight gain in HFD-fed mice but failed to suppress the development of obesity in leptin ob–mutant (Lepob-mutant) mice and adenylate cyclase 3–mutant (Adcy3-mutant) mice on a chow diet. Our data reveal that GPR75 is a ciliary protein expressed in the brain and plays an important role in regulating food intake.
Yiao Jiang, Yu Xun, Zhao Zhang
Severe viral pneumonia can induce rapid expansion of KRT5+ basal-like cells in small airways and alveoli; this forms a scar-like structure that persists in the injured alveoli and impedes normal alveolar epithelium regeneration. In this study, we investigated the mechanism by which viral infection induced this remodeling response. Through comparing different lung-injury models, we demonstrated that infection induced strong IFN-γ signal–stimulated dysplastic KRT5+ cell formation. Inactivation of interferon receptor 1 (Ifngr1) reduced dysplastic cell formation, ameliorated lung fibrosis, and improved lung-function recovery. Mechanistically, IFN-γ regulated dysplastic cell formation via the focal adhesion kinase (FAK)/Yes-associated protein 1 (YAP) pathway. Inhibiting FAK/Src diminished IFN-γ–induced YAP nuclear translocation and dysplastic cell formation. Inhibiting YAP during viral infection prevented dysplastic cell formation, whereas inhibiting YAP in persistent KRT5+ cells led to their conversion into distal club cells. Importantly, human dysplastic cells exhibited elevated FAK and YAP activity, and IFN-γ treatment promoted the transformation of human alveolar progenitor cells into dysplastic cells. These findings uncover the role of infection-induced inflammatory response in alveolar remodeling and may provide potential therapeutic avenues for the treatment of alveolar remodeling in patients with severe viral pneumonia.
Xiuyu Lin, Weicheng Chen, Guilin Yang, Jiazhu Zhang, Huilin Wang, Zeyu Liu, Ying Xi, Tao Ren, Bo Liu, Pengfei Sui
During antiretroviral therapy (ART), most people living with HIV-1 have undetectable HIV-1 RNA in their plasma. However, they occasionally present with new or progressive neurologic deficits and detectable HIV-1 RNA in the cerebrospinal fluid (CSF), a condition defined as neurosymptomatic HIV-1 CSF escape (NSE). We explored the source of neuropathogenesis and HIV-1 RNA in the CSF during NSE by characterizing HIV-1 populations and inflammatory biomarkers in CSF from 25 individuals with NSE. HIV-1 populations in the CSF were uniformly drug resistant and adapted to replication in CD4+ T cells, but differed greatly in genetic diversity, with some having low levels of diversity similar to those observed during untreated primary infection and others having high levels like those during untreated chronic infection. Higher diversity in the CSF during NSE was associated with greater CNS inflammation. Finally, optimization of ART regimen was associated with viral suppression and improvement of neurologic symptoms. These results are consistent with CNS inflammation and neurologic injury during NSE being driven by replication of partially drug-resistant virus in CNS CD4+ T cells. This is unlike nonsuppressible viremia in the plasma during ART, which typically lacks clinical consequences and is generated by virus expression without replication.
Laura P. Kincer, Ameet Dravid, Mattia Trunfio, Andrea Calcagno, Shuntai Zhou, Riccardo Vercesi, Serena Spudich, Magnus Gisslen, Richard W. Price, Paola Cinque, Sarah B. Joseph
Infants with biallelic IL7R loss-of-function variants have severe combined immune deficiency (SCID) characterized by the absence of autologous T lymphocytes, but normal counts of circulating B and NK cells (T–B+NK+ SCID). We report 6 adults (aged 22 to 59 years) from 4 kindreds and 3 ancestries (Colombian, Israeli Arab, Japanese) carrying homozygous IL7 loss-of-function variants resulting in combined immunodeficiency (CID). Deep immunophenotyping revealed relatively normal counts and/or proportions of myeloid, B, NK, and innate lymphoid cells. By contrast, the patients had profound T cell lymphopenia, with low proportions of innate-like adaptive mucosal-associated invariant T and invariant NK T cells. They also had low blood counts of T cell receptor (TCR) excision circles, recent thymic emigrant T cells and naive CD4+ T cells, and low overall TCR repertoire diversity, collectively indicating impaired thymic output. The proportions of effector memory CD4+ and CD8+ T cells were high, indicating IL-7–independent homeostatic T cell proliferation in the periphery. Intriguingly, the proportions of other T cell subsets, including TCRγδ+ T cells and some TCRαβ+ T cell subsets (including Th1, Tfh, and Treg) were little affected. Peripheral CD4+ T cells displayed poor proliferation, but normal cytokine production upon stimulation with mitogens in vitro. Thus, inherited IL-7 deficiency impairs T cell development less severely and in a more subset-specific manner than IL-7R deficiency. These findings suggest that another IL-7R–binding cytokine, possibly thymic stromal lymphopoietin, governs an IL-7–independent pathway of human T cell development.
Carlos A. Arango-Franco, Masato Ogishi, Susanne Unger, Ottavia M. Delmonte, Julio César Orrego, Ahmad Yatim, Margarita M. Velasquez-Lopera, Andrés F. Zea-Vera, Jonathan Bohlen, Marwa Chbihi, Antoine Fayand, Juan Pablo Sánchez, Julian Rojas, Yoann Seeleuthner, Tom Le Voyer, Quentin Philippot, Kathryn J. Payne, Adrian Gervais, Lucia V. Erazo-Borrás, Luis A. Correa-Londoño, Axel Cederholm, Alejandro Gallón-Duque, Pedro Goncalves, Jean-Marc Doisne, Liran Horev, Bénédicte Charmeteau-de Muylder, Jesús Á. Álvarez, Diana M. Arboleda, Lizet Pérez-Zapata, Estefanía Vásquez-Echeverri, Marcela Moncada-Vélez, Juan A. López, Yolanda Caicedo, Boaz Palterer, Pablo J. Patiño, Carlos J. Montoya, Matthieu Chaldebas, Peng Zhang, Tina Nguyen, Cindy S. Ma, Mohamed Jeljeli, Juan F. Alzate, Felipe Cabarcas, Taushif Khan, Darawan Rinchai, Jean-Luc Prétet, Bertrand Boisson, Generalized Verrucosis Japanese Consortium, Nico Marr, Ruba Ibrahim, Vered Molho-Pessach, Stéphanie Boisson-Dupuis, Dimitra Kiritsi, João T. Barata, Nils Landegren, Bénédicte Neven, Laurent Abel, Andrea Lisco, Vivien Béziat, Emmanuelle Jouanguy, Jacinta Bustamante, James P. Di Santo, Stuart G. Tangye, Luigi D. Notarangelo, Rémi Cheynier, Ken Natsuga, Andrés A. Arias, José Luis Franco, Klaus Warnatz, Jean-Laurent Casanova, Anne Puel
NKT cells are innate-like T cells, recruited to the skin during viral infection, yet their contributions to long-term immune memory to viruses are unclear. We identified granzyme K, a product made by cytotoxic cells including NKT cells, as linked to induction of Th1-associated antibodies during primary dengue virus (DENV) infection in humans. We examined the role of NKT cells in vivo using DENV-infected mice lacking CD1d-dependent (CD1ddep) NKT cells. In CD1d-KO mice, Th1-polarized immunity and infection resolution were impaired, which was dependent on intrinsic NKT cell production of IFN-γ, since it was restored by adoptive transfer of WT but not IFN-γ–KO NKT cells. Furthermore, NKT cell deficiency triggered immune bias, resulting in higher levels of Th2-associated IgG1 than Th1-associated IgG2a, which failed to protect against a homologous DENV rechallenge and promoted antibody-dependent enhanced disease during secondary heterologous infections. Similarly, Th2 immunity, typified by a higher IgG4/IgG3 ratio, was associated with worsened human disease severity during secondary infections. Thus, CD1ddep NKT cells establish Th1 polarity during the early innate response to DENV, which promotes infection resolution, memory formation, and long-term protection from secondary homologous and heterologous infections in mice, with consistent associations observed in humans. These observations illustrate how early innate immune responses during primary infections can influence secondary infection outcomes.
Youngjoo Choi, Wilfried A.A. Saron, Aled O’Neill, Manouri Senanayake, Annelies Wilder-Smith, Abhay P.S. Rathore, Ashley L. St. John
The study of transcription factors that determine specialized neuronal functions has provided invaluable insights into the physiology of the nervous system. Peripheral chemoreceptors are neurone-like electrophysiologically excitable cells that link the oxygen concentration of arterial blood to the neuronal control of breathing. In the adult, this oxygen chemosensitivity is exemplified by type I cells of the carotid body, and recent work has revealed one isoform of the hypoxia-inducible transcription factor (HIF), HIF-2α, as having a nonredundant role in the development and function of that organ. Here, we show that activation of HIF-2α, including isolated overexpression of HIF-2α but not HIF-1α, is sufficient to induce oxygen chemosensitivity in adult adrenal medulla. This phenotypic change in the adrenal medulla was associated with retention of extra-adrenal paraganglioma-like tissues resembling the fetal organ of Zuckerkandl, which also manifests oxygen chemosensitivity. Acquisition of chemosensitivity was associated with changes in the adrenal medullary expression of gene classes that are ordinarily characteristic of the carotid body, including G protein regulators and atypical subunits of mitochondrial cytochrome oxidase. Overall, the findings suggest that, at least in certain tissues, HIF-2α acts as a phenotypic driver for cells that display oxygen chemosensitivity, thus linking 2 major oxygen-sensing systems.
Maria Prange-Barczynska, Holly A. Jones, Yoichiro Sugimoto, Xiaotong Cheng, Joanna D.C.C. Lima, Indrika Ratnayaka, Gillian Douglas, Keith J. Buckler, Peter J. Ratcliffe, Thomas P. Keeley, Tammie Bishop
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